Heat stress is one of the most important issues in broiler flocks impairing animal health and productivity. On a cellular level, excess heat exposure can trigger heat shock response acting for the restoration of cell homeostasis by several mechanisms, such as affecting heat shock protein synthesis, redox homeostasis and pro-inflammatory cytokine production. The major aim of this study was to establish a novel avian hepatocyte—nonparenchymal cell co-culture as a model for investigating the cellular effects of heat stress and its interaction with inflammation in chicken liver. Cell fractions were isolated by differential centrifugation from a freshly perfused chicken liver, and hepatocyte mono-cultures as well as hepatocyte–nonparenchymal cell co-cultures (with cell ratio 6:1, hepatocytes to nonparenchymal cells, mimicking a milder hepatic inflammation) were prepared. Isolated and cultured cells were characterized by flow cytometry and immunocytochemistry applying hepatocyte- and macrophage-specific antibodies. Confluent cell cultures were exposed to 43 °C temperature for 1 or 2 h, while controls were cultured at 38.5 °C. The metabolic activity, LDH enzyme activity, reactive oxygen species (H2O2) production, extracellular concentration of heat shock protein 70 (HSP70), and that of the pro-inflammatory cytokines interleukin (IL-)6 and IL-8 were assessed. Shorter heat stress applied for 1 h could strongly influence liver cell function by significantly increasing catabolic metabolism and extracellular H2O2 release, and by significantly decreasing HSP70, IL-6, and IL-8 production on both cell culture models. However, all these alterations were restored after 2 h heat exposure, indicating a fast recovery of liver cells. Hepatocyte mono-cultures and hepatocyte—nonparenchymal cell co-cultures responded to heat stress in a similar manner, but the higher metabolic rate of co-cultured cells may have contributed to a better capability of inflamed liver cells for accommodation to stress conditions. In conclusion, the established new primary cell culture models provide suitable tools for studying the hepatic inflammatory and stress response. The results of this study highlight the impact of short-term heat stress on the liver in chickens, underline the mediatory role of oxidative stress in acute stress response, and suggest a fast cellular adaptation potential in liver cells.

The intestinal microbiome can influence the efficiency and the health status of its host’s digestive system. Indigestible non-starch polysaccharides (NSP) serve as substrates for bacterial fermentation, resulting in short-chain fatty acids like butyrate. In broiler’s nutrition, dietary crude protein (CP) and butyrate’s presence is of particular interest for its impact on intestinal health and growth performance. In this study, we evaluated the effect on the microbial ecology of the ceca of dietary supplementations, varying the cereal type (maize and wheat), adequate levels of CP and supplementation of sodium butyrate on broiler chickens with 21 days. The overall structure of bacterial communities was statistically affected by cereal type, CP, and sodium butyrate (p = 0.001). Wheat in the diet promoted the presence of Lactobacillaceae, Bifidobacteriaceae and Bacteroides xylanisolvens, which can degrade complex carbohydrates. Maize positively affected the abundance of Bacteroides vulgatus. The addition of CP promoted the family Rikenellaceae, while sodium butyrate as feed supplement was positively related to the family Lachnospiraceae. Functional predictions showed an effect of the cereal type and a statistical significance across all supplementations and their corresponding interactions. The composition of diets affected the overall structure of broilers’ intestinal microbiota. The source of NSP as a substrate for bacterial fermentation had a stronger stimulus on bacterial communities than CP content or supplementation of butyrate.

Trichothecene mycotoxins such as T-2 toxin cause severe problems for agriculture, as well as for veterinary medicine. As liver is one of the key organs in metabolism, the main aim of our study was to investigate the immunomodulatory and cytotoxic effects of T-2 toxin, using primary hepatocyte mono-culture and hepatocyte—nonparenchymal cell (predominantly Kupffer cell) co-culture models of chicken. Cultures were exposed to 10 (T10 group), 100 (T100 group) and 1000 (T1000 group) nmol/L T-2 toxin treatment for 8 or 24 h. Alterations of cellular metabolic activity, the production of reactive oxygen species (extracellular H2O2), heat shock protein 70 (HSP70), and the concentration of different inflammatory cytokines such as interleukin (IL-)6 and IL-8 were investigated. Metabolic activity was intensely decreased by T-2 toxin administration in all of the cell culture models, in every applied concentration and incubation time. Concentrations of HSP70 and IL-8 were significantly increased in hepatocyte mono-cultures exposed to higher T-2 toxin levels (both in T100 and T1000 groups for HSP70 and in T1000 group for IL-8, respectively) compared to controls after 24 h incubation. Similarly, IL-6 levels were also significantly elevated in the T100 and T1000 groups in both of mono- and co-cultures, but only after 8 h of incubation time. In spite of the general harmful effects of T-2 toxin treatment, no significant differences were observed on reactive oxygen species production. Furthermore, the two cell culture models showed different levels of H2O2, HSP70, and IL-8 concentrations independently of T-2 toxin supplementation. In conclusion, the established primary cell cultures derived from chicken proved to be proper models to study the specific molecular effects caused by T-2 toxin. Metabolic activity and immune status of the different examined cell cultures were intensively affected; however, no changes were found in H2O2 levels.

Short chain fatty acid (SCFA) butyrate has various beneficial effects on the gut microbiota as well as on the overall health status and metabolism of the host organism. The modulatory role of butyrate on gut barrier integrity reflected by tight junction protein expression has been already described in mammalian species. However, there is limited information available regarding chickens. Therefore, the main aim of this study was to monitor the effects of protected butyrate on claudin barrier protein and monocarboxylate transporter 1 abundance in different gastrointestinal segments of chickens as well as the growth performance of broiler chickens. The effect of protected butyrate on the caecal microbiota was monitored by quantifying the concentrations of total eubacteria and key enzymes of butyrate production. Furthermore, intestinal SCFA concentrations were also measured. Based on the data obtained, protected butyrate increased the overall performance as well as the barrier integrity of various gut segments. Protected butyrate also positively affected the SCFA concentration and composition. These findings provide valuable insight into the complex effects of protected butyrate on broiler gut health, highlighting the beneficial effects in improving intestinal barrier integrity and performance parameters.

The liver with resident tissue macrophages is the site of vivid innate immunity, activated also by pathogen-associated molecular patterns (PAMPs) leaking through the intestinal barrier. As gut-derived inflammatory diseases are of outstanding importance in broiler chickens, the present study aimed to establish a proper hepatic inflammatory model by comparing the action of different PAMPs from poultry pathogens on chicken 2D and 3D primary hepatocyte—non-parenchymal cell co-cultures, the latter newly developed with a magnetic bioprinting method. The cultures were challenged by the bacterial endotoxins lipopolysaccharide (LPS) from Escherichia coli, lipoteichoic acid (LTA) from Staphylococcus aureus and by enterotoxin (ETxB) from Escherichia coli, Salmonella Typhimurium derived flagellin, phorbol myristate acetate (PMA) as a model proinflammatory agent and polyinosinic polycytidylic acid (poly I:C) for mimicking viral RNA exposure. Cellular metabolic activity was assessed with the CCK-8 test, membrane damage was monitored with the lactate dehydrogenase (LDH) leakage assay and interleukin-6 and -8 (Il-6 and -8) concentrations were measured in cell culture medium with a chicken specific ELISA. Both LPS and LTA increased the metabolic activity of the 3D cultures, concomitantly decreasing the LDH leakage, while in 2D cultures ETxB stimulated, PMA and poly I:C depressed the metabolic activity. Based on the moderately increased extracellular LDH activity, LTA seemed to diminish cell membrane integrity in 2D and poly I:C in both cell culture models. The applied endotoxins remarkably reduced the IL-8 release of 3D cultured cells, suggesting the effective metabolic adaptation and the presumably initiated anti-inflammatory mechanisms of the 3D spheroids. Notwithstanding that the IL-6 and IL-8 production of 2D cells was mostly not influenced by the endotoxins used, only the higher LTA dose was capable to evoke an IL-8 surge. Flagellin, PMA and poly I:C exerted proinflammatory action in certain concentrations in both 2D and 3D cultures, reflected by the increased cellular IL-6 release. Based on these data, LTA, flagellin, PMA and poly I:C can be considered as potent candidates to induce inflammation in chicken primary hepatic cell cultures, while LPS failed to trigger proinflammatory cytokine production, suggesting the relatively high tolerance of avian liver cells to certain bacterial endotoxins. These results substantiate that the established 3D co-cultures seemed to be proper tools for testing potential proinflammatory molecules; however, the remarkable differences between 2D and 3D models should be addressed and further studied.